This is a competing renewal application for R01 GM066047 entitled Chemical Glycoproteomics. The broad objective of this project, now it its 12th year, is to develop chemical tools for studying mucin-type O- glycosylation and the polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) that initiate this widespread posttranslational modification. Mucin-type O-glycosylation has proven far more difficult to study than other prevalent forms, such as N-glycosylation and O-GlcNAcylation. Our parts list of proteins and sites modified with O-glycans is far from complete, and we have little information regarding the biological substrates of the 20 human ppGalNAcTs. Yet, genetic studies have linked dysregulated expression of ppGalNAcTs to many human diseases, and mutations in O-glycosites can lead to profound pathologies. Thus there is a need for new tools that accelerate research into the molecular basis and biological significance of mucin-type O- glycosylation. This project builds on the work from previous granting periods with three Specific Aims. In Aim 1, we will develop a glycoproteomics platform for identifying O-glycosylated proteins and mapping O-glycosites. We will employ metabolic labeling with an azide-functionalized GalNAc derivative (GalNAz) followed by click chemistry with biotin probes for selective enrichment of mucin-type O-glycoproteins. After trypsinolysis, glycosylated peptides will be identified and sequenced using our mass-independent IsoStamp technology, developed in the previous funding period. The glycoproteomics platform will be used to identify O-glycoproteins and map O- glycosites from cancerous and normal human prostate tissues, with the long-term goal of biomarker discovery. In Aim 2, we will develop a chemical genetic approach for identifying biological substrates of ppGalNAcT family members. We will employ the bump-hole strategy to orthogonalize individual ppGalNAcTs so they can uniquely mark their cellular substrates with azidosugars. The azide-modified glycoproteins and glycosites will be identified using the glycoproteomics platform from Aim 1. Finally, in Aim 3 we will identify biological substrates of ppGalNAcT-7, the expression of which is correlated with tumor progression. Using the tools of Aims 1 and 2, we will map O-glycosites initiated by ppGalNAcT-7 in cervical cancer cells. This dataset will fuel hypotheses regarding the enzyme's role in disease progression.